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Online-Only Supplement 1  

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Vasculoprotective effects of dietary cocoa flavanols in hemodialysis patients: a 3  

double-blind, randomized, placebo-controlled trial 4  

5  

Tienush Rassaf, MD1*; Christos Rammos, MD1*; Ulrike-B. Hendgen-Cotta, PhD1; 6  

Christian Heiss, MD1; Werner Kleophas, MD2; Frank Dellanna, MD2; Jürgen. Floege, MD3; 7  

Gerd R. Hetzel, MD2; Malte Kelm, MD1,4 8  

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Methods 1  

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For the determination of vascular functions (FMD and PWV) participants were required 3  

to fast at least 5 hours prior to the measurements. Comparisons using area-under the curve 4  

were conducted using Prism 5.0 (GraphPad) calculating the total peak area of FMD for each 5  

timepoint assessed (before, during and after HD) for the acute and acute-on chronic study, 6  

respectively. 7  

Brachial blood pressure (BP) was measured in duplicate in the non-fistula arm by cuff 8  

and mercury sphygmomanometer after the participant had rested in a seated position for 10 9  

min and the average of the 2 measurements was recorded. 10  

After the participants had rested for 10 minutes we determined arterial stiffness by 11  

PWV. PWV was calculated from sequential recordings of electrocardiogram referenced 12  

carotid and femoral pressure waveforms obtained by using tonometry with the SphygmoCor 13  

device and transducer (AtCor Medical, Sydney, Australia). Wave transit-time was determined 14  

using the distance between carotid and femoral sites estimated from the distance between 15  

each artery location, the sternal notch and the R-wave of a simultaneously recorded 16  

electrocardiogram as reference frame (1). 17  

Carotid intima-media thickness (IMT) was determined using high-resolution ultrasound 18  

(Vivid i ultrasound, GE Healthcare, Munich Germany). IMT was measured 1 cm distal the 19  

carotid bifurcation using end-diastolic (minimum dimension) images of the far wall of the 20  

distal common carotid artery. 21  

Blood was drawn for clinical routine and the Institute of Clinical Chemistry and 22  

Laboratory Diagnostics, University Hospital Duesseldorf performed all analyses unless noted 23  

otherwise. (-)-Epicatechin and its related metabolites in plasama and dialysate fluid were 24  

determined by HPLC-FLD/UV and electrochemical detection using authentic standards 25  

provided by Mars Symbioscience, as described (2). Plasma nitrite and nitrate were 26  

  3  

determinded by HPLC technique (Eicom, Eno-20), as described (3,4). The advanced 1  

glycation end product Carboxymethyl Lysine (CML, microcoat, Bernried, Germany), markers 2  

of inflammation hs-Interleukin 6 (IL-6, eBioscience, Vienna, Austria) and oxidative Stress 3  

oxidized LDL, OxLDL, Mercodia, Uppsala, Sweden) were measured by ELISAs following 4  

the manufacturers protocol, respectively. 5  

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Tables 1  

Supplemental Table 1: Inclusion and Exclusion Criteria. 2  

 3  Inclusion Criteria: Exclusion Criteria  

end stage renal disease acute renal failure

>18 years acute infection

chronic intermittent hemodialysis heart failure (NYHA IV)

pregnancy

anuria

 4   5  

  5  

1  

Supplemental Table 2: Test drink composition and nutritional information. 2  

3  

Total Daily Intake CF Placebo

Total cocoa flavanols (mg) 900 ND

(-)-Epicatechin 128 ND

(+)-Catechin 4 ND

(-)-Catechin 14 ND

(+)-Epicatechin ND ND

Total calories (kcal) 50 50

Total fat (g) 0 0

Stearic acid (g) 0 0

Total carbohydrates (g) 12 12

Sugars (g) 0.4 0.2

Protein (g) 0.2 0.2

Fiber (g) 1.2 0.8

Sodium (mg) 6 6

Potassium (mg) 190 170

Magnesium (mg) 2 2

Caffeine (mg) 20 12

Theobromine (mg) 88 92

4  

ND=Not detectable 5  

  6  

Supplemental Table 3: Baseline patient characteristics of the acute study. 1  

2  

n 10

Male n 9

Age (years) 64.1 ± 9.5

Weight (kg) 87.9 ± 9.9

Body mass index (kg/m2) 28.9 ± 1.4

Renal diagnosis (n)

Hypertensive/large vessel 2

Diabetic nephropathy 3

Polycystic kidney disease 1

Glomerulonephritis 2

Other/miscellaneous 2

Dialysis vintage (m) 16 (12, 46)

Hypertension (n) 10

Diabetes (n) 4

Current smoker (n) 3

Hypercholesterinaemia (n) 3

CVD (n) 2

Medication (n) ASS 5

Statin 6

AT blocker 3

ACE-I 4

b blocker 6

Ca-channel blocker 2

Diuretics 10

 3    4  

  7  

Supplemental Figure 1 1  

 2  

3    4   5   6  

Placebo Flavanols0

5

10

15

NM

D (%

)

  8  

1  

Supplemental Figure 3. 2  

3  

4  

5  

pre post pre post4.0

4.5

5.0

5.5

6.0

Placebo Flavanols

Pota

ssiu

m (m

mol

/l)

pre post pre post0

50

100

150

Placebo Flavanols

MBP

(mm

Hg)

pre post pre post7.20

7.25

7.30

7.35

7.40

Placebo Flavanols

pH

pre post pre post0

20

40

60

80

100

Placebo Flavanols

Hea

rt ra

te (b

pm)

A B

C D

  9  

Supplemental Figure 3. 1  

 2    3  

4   5  

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1  

Supplemental Figure 4. 2  

3   4  

Day 0 Day 30 Day 0 Day 300

2

4

6

Placebo Flavanols

FMD

(%)

*

  11  

Supplemental Figure 5. 1  

2  

3   4   5     6  

Placebo Flavanol0

5

10

15

NM

D (%

)

  12  

Figure Legends 1  

 2  Supplemental Figure 1. Acute study: Endothelial independent function in patients with 3  

ESRD. 4  

No effect for Nitroglycerin-mediated vasodilation (NMD) i.e., endothelium-independent 5  

vasodilation in ESRD patients following acute ingestion of CF or placebo, n=5. 6  

7  

Supplemental Figure 2. Baseline acute study: Acute effect of dietary flavanols on clinical 8  

routine and hemodynamics in patients with ESRD. 9  

No effect on potassium and pH values (A) and (B) or mean blood pressure (MBP) and heart 10  

rate (C) and (D) in CF group vs. placebo. *p<0.05, n=10, Data are given as mean ± SEM. 11  

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Supplemental Figure 3. Baseline acute study: Plasma epicatechin metabolites in ESRD 13  

after a singular ingestion of flavanols. 14  

Plasma concentration of structurally-related epicatechin metabolites (SREM) in end-stage 15  

renal disease patients, with CF ingestion at 0 hr. SREM quantified corresponds to epicatechin-16  

3′-b-D-glucuronide (E3G), (−)-epicatechin-3′­sulfate (E3S), (−)-epicatechin-5­sulfate (E5S), 17  

3′­O­methyl(−)-epicatechin-5­sulfate (3MES), 3′­O­methyl(−)-epicatechin-7­sulfate 18  

(3ME7S), 4′­O­methyl(−)-epicatechin-5-sulfate (4ME5S) and 4′­O­methyl(−)-epicatechin-7-19  

sulfate (4ME7S). Data are expressed in nM (nmol of SREM/L of plasma). *denotes p<0.05 20  

compared to baseline, ∆ compared to 1 hr value after CF ingestion, n=10. 21  

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Supplemental Figure 4. Chronic study: Chronic effects of dietary flavanols in patients 1  

with ESRD. 2  

Chronic effect on endothelial function following a 30-day ingestion period of CF or placebo 3  

(FMD, flow-mediated dilation). *denotes p<0.05 4  

5  

Supplemental Figure 5. Chronic study: Endothelial independent function in patients 6  

with ESRD. 7  

No effect for Nitroglycerin-mediated vasodilation (NMD) i.e., endothelium-independent 8  

vasodilation in ESRD patients following chronic ingestion of CF or placebo, n=7-9. 9  

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References 1  

1. Rammos C, Hendgen-Cotta UB, Sobierajski J, Adamczyk S, Hetzel GR, Kleophas W, Dellanna F, 2  Kelm M, Rassaf T: Macrophage migration inhibitory factor is associated with vascular dysfunction in 3  patients with end-stage renal disease. Int J Cardiol 168: 5249-5256, 2013 4  

2. Ottaviani JI, Momma TY, Kuhnle GK, Keen CL, Schroeter H: Structurally related (-)-epicatechin 5  metabolites in humans: assessment using de novo chemically synthesized authentic standards. Free 6  Radic Biol Med 52: 1403-1412, 2012 7  

3. Hendgen-Cotta UB, Luedike P, Totzeck M, Kropp M, Schicho A, Stock P, Rammos C, Niessen M, 8  Heiss C, Lundberg JO, Weitzberg E, Kelm M, Rassaf T: Dietary nitrate supplementation improves 9  revascularization in chronic ischemia. Circulation 126: 1983-1992, 2012 10  

4. Rammos C, Hendgen-Cotta UB, Sobierajski J, Bernard A, Kelm M, Rassaf T: Dietary nitrate reverses 11  vascular dysfunction in older adults with moderately increased cardiovascular risk. J Am Coll Cardiol 12  63: 1584-1585, 2014 13  

14  1. Rammos C, Hendgen-Cotta UB, Sobierajski J et al. Macrophage migration inhibitory factor is 15  

associated with vascular dysfunction in patients with end-stage renal disease. Int J Cardiol. 16  2013;168(6):5249-5256. 17  

2. Ottaviani JI, Momma TY, Kuhnle GK, Keen CL, Schroeter H. Structurally related (-)-epicatechin 18  metabolites in humans: assessment using de novo chemically synthesized authentic standards. Free 19  Radic Biol Med. 2012;52(8):1403-1412. 20  

3. Hendgen-Cotta UB, Luedike P, Totzeck M et al. Dietary nitrate supplementation improves 21  revascularization in chronic ischemia. Circulation. 2012;126(16):1983-1992. 22  

4. Rammos C, Hendgen-Cotta UB, Sobierajski J et al. Dietary nitrate reverses vascular dysfunction in 23  older adults with moderately increased cardiovascular risk. J Am Coll Cardiol. 2014;63(15):1584-1585. 24  

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